Tresulphan, a human carcinogen, has been reported to induce chromosomal aberrations in plants, mutations in Salmonella, and micronuclei in mouse bone marrow. The biological effects of Treosulphan (toxicity, mutagenicity, etc.) were postulated to be mediated by its hydrolysis product, dl-1.2:3.4-diepoxybutane (DEB). In the present investigation, the cytotoxicity and mutagenicity of Tresulphan and DEB were studied in AS52 cells, a PSV2gpt-transformed Chinese hamster ovary (CHO) cell line, by measuring the colony forming efficiency (CFE) and mutant frequency (MF). Factors such as the pH of the treatment medium, the duration of exposure, and cell density during the treatments, were also investigated. Treosulphan (0.1-1.0 milli-M) was toxic and mutagenic, causing a significant dose-related decrease in CFE and increased MF, which, within certain ranges, were related pH and time of exposure. DEB was also cytctcxic and mutagenic of a much lower dose (0.025 mM). Its cytotoxicity and mutagenicity were not affected by pH. Responses to the toxicity and mutagenicity of both Treosulphan and DEB were increasingly modulated by cell density during the exposure. These results suggest that the biological effects of Treosulphan are mediated by its hydrolysis product, DEB, and the biotransformation of Treosulphan to DEB was highly dependent upon the pH of the treatment medium and the time of treatment. Studies are underyway to evaluate the chromosome damaging ability of Treosulphan. Mutants induced by both chemicals will be sequenced to determine if each produces the same ultimate mutagen.